We have shown that the levels of nitrite in platelets during storage in vitro drop slightly and if this is due to NO formation may contribute to keeping the platelets functional for transfusion.In the last two years we have shown that at physiological nitrite concentrations in the presence of partially deoxygenated red blood cells, human platelet reactivity, as measured by aggregometry or cell surface makers, in response to various agonists, including collagen, ADP and thrombin, is inhibited. This effect is maximal at about 20% hematocrit and is quite marked at levels of 1 microM nitrite and correlates with changes in CGMP levels. Almost certainly it is due to the formation of NO from nitrite by reduction by the red cell intracellular hemoglobin and has been interpreted as the best evidence that NO formed in this way can exit the erythrocyte. We have demonstrated the same effects in rodent platelets, where we can increase or decrease plasma nitrite levels by diet, genetic manipulation, or the use of pharmacological agents. Bleeding time is also inhibited in these animals by nitrite. We have also shown that these effects can be measured by flow cytometry of platelet surface markers. More recently we have used thromboelastometry, with the TEG machine to demonstrate these effects in human platelets with an assay that is now more widely used clinically. We have begun to work with several physiology groups to test these effects in vascular flow models. Our current hypothesis is that they contribute to differences between venous and arterial blood clots and may have pharmacological potential to prevent blood clotting. For some years we have been working with a group in Lisbon to develop electrode-based assays for nitrite in water and other environmental fluids; in view of the importance of nitrite in blood and other biological fluids we have now redirected this work to a more micro scale so that measurements on animal and human samples may be feasible. Detailed metabolic studies of nitrate-nitrite-NO metabolism in animals and people require tracer studies which may be approached using non-toxic heavy isotopes. Our collaborators in Newcastle are developing protocols to do this in animals and then perhaps in human subjects. Lastly our collaborators in Sao Paulo, Brazil have much experience in pharmacology of NO and we are working with them on various animal models of hypertension with the hope of eventual clinical studies.